Autophagy is a conserved homeostatic process by which cells degrade and recycle cytoplasmic content and organelles. Recent work has identified a critical role for autophagy in host defense against intracellular bacteria, viruses, and parasites. Thus, new therapeutic approaches aimed at augmenting host autophagy bear potential to combat a diversity of human pathogens for which current treatment strategies are inadequate. While significant progress has been made in identifying and defining the roles of autophagy related (ATG) genes, it is clear that they are insufficient to account for the exquisite regulation of microbialhost cell interactions. The overarching focus of this application is to (1) define a systems-level understanding of autophagy by identifying genes and pathways that are essential for antibacterial autophagy and (2) leverage these insights to develop/test lead compounds that enhance autophagy and function as broadspectrum anti-infective agents. In Aim 1, we will identify candidate host genes and pathways that regulate and serve as effectors in antibacterial autophagy using a genome-wide siRNA screen. These experiments include screening for genes that affect autophagy of NIAID Category B/C pathogens: Salmonella Typhimurium, Mycobacterium tuberculosis, and Listeria monocytogenes. In Aim 2, we will identify the mechanism of action of candidate genes that function as effectors and/or regulators of antibacterial autophagy; this work will identify/prioritize new target pathways in which to screen for small molecules that augment autophagy. In Aim 3, we will determine the efficacy of existing (and future) lead compounds/peptides as broad-spectrum anti-infective agents in models of bacterial infection. Collectively, these studies will provide insights into key cellular mechanisms that regulate antibacterial autophagy and facilitate the development of new therapeutic approaches for the treatment of high priority infectious diseases. Design, execution and analysis of experiments will require input from Core B. Moreover, through interactions with the other projects in this proposal (led by Drs. Levine, Schreiber, and Virgin), we expect these efforts to produce an autophagy connectivity network integrated across multiple pathogens and to identify lead compounds with broad-spectrum anti-infective activity.